Fungi remain a medically, industrially and agriculturally important group of organisms. Opportunistic pathogens such as Candida albicans and Aspergillus fumigatus can cause serious infections in humans. Recent increases in the incidence of infection of immunocomprised individuals by fungal pathogens has stimulated efforts to develop more effective antifungal therapeutic agents. There is a long history of the use of fungi in the food industry and for the production of biologicals, organic acids and pharmaceutical intermediates. The ability to genetically alter yeast and filamentous fungi by DNA-mediated transformation has considerably increased their industrial potential. Despite impressive progress in the development of chemical fungicides for agricultural application, yield losses due to plant pathogenic fungi remains a significant problem worldwide. The wide spread use of chemical fungicides is also complicated by the persistent development of acquired resistance and by a negative environmental impact.
Few plant disease epidemics have received as much attention or generated as much consternation as the chestnut blight epidemic that occurred in North America and Europe early this century as a result of the unintentional introduction of the Asian fungus, Cryphonectria (Endothia) parasitica. Contributing factors include the magnitude of the disease, e.g., an estimated 3.5 billion trees were destroyed in the North American eastern hardwood forest by 1950; the aesthetic, ecological and economic value of the host; and the total inability to devise effect control measures (Anagnostakis, 1982; Roane et al., 1986; Griffin 1986; MacDonald and Fulbright 1991). Consequently reports in the 1960's and 1970's of natural and artificial biological control of chestnut blight due to the phenomenon of "transmissible hypovirulence" generated considerable interest (Grente, 1965; Grente and Berthelay-Sauret, 1978; Van Alfen et al., 1975). Natural variants of C. parasitica that exhibited reduced levels of virulence (hypovirulent) were first discovered in Italy and were later identified in different geographic locations in Europe and North America (Grente and Berthelay-Sauret, 1978; Anagnostakis, 1982; MacDonald and Fulbright, 1991). While virulent C. parasitica strains penetrate and destroy bark and cambium layers causing wilting and death, hypoviolent strains usually produce superficial cankers that eventually heal as a result of host defense mechanisms. The basis for disease control lies with the ability of the hypovirulence phenotype to be transmitted to virulent strains resulting in conversion of the recipient to hypovirulence (Grente, 1965; Grente and Berthelay-Sauret, 1978; Van Alfen et al., 1975). Studies with auxotrophic mutant strains indicated that the genetic element responsible for the hypovirulence phenotype corresponded to a cytoplasmic determinant that was transferred by hyphal anastomosis (fusion of hyphae) (Van Alfen et al., 1975). Day et al. (1977) subsequently reported that hypovirulent C. parasitica strains contained double-stranded (ds) RNA species that were generally absent in virulent C. parasitica strains. Moreover, these ds RNAs were observed to be transmitted coincidentally with the hypovirulence phenotype during anastomosis.
Recent characterizations of structural and functional properties of hypovirulence-associated dsRNAs have provided evidence for a viral origin even though these RNAs appear not to have be encapsidated within a discrete virus particle. For example, the large dsRNA, L-dsRNA, present in hypovirulent C. parasitica strain EP713 was shown to encode two large polyproteins that undergo autoproteolytic processing during translation (Choi et al., 1991a; 1991b; Shapira et al., 1991a; Shapira and Nuss, 1991). Recent computer-assisted analysis of the predicted amino acid sequences of these polyproteins revealed five distinct domains with significant sequence similarity to previously described conserved domains within protein products encoded by members of the potyvirus group of positive-strand RNA plant viruses (Koonin et al., 1991). Phylogenic trees, derived from the alignment of one specific domain, that of the putative RNA-dependent RNA polymerase, with the sequences of all known viral RNA polymerases strongly suggested that L-dsRNA and potyvirus genomes share a common ancestry. Based on the similarity of the L-dsRNA genetic organization and expression strategy to those of several viral genomes and the apparent evolutionary relationship to the potyviruses, the term hypovirulence-associated virus (HAV) will be used to denote this class of genetic element (Shapira et al., 1991).
Direct analysis of the L-dsRNA present in hypovirulent C. parasitica strain EP713 has shown that one strand contains a 3'-poly (A) tail that is base paired to a stretch of poly (U) present at the 5'-terminus of the complementary strand (Hiremath et al., 1986). Sequence analysis of multiple cDNA clones that spanned the entire length of L-dsRNA revealed that the molecule consists of 12,712 bp, excluding the poly(A):poly(U) homopolymer domain, and that only the poly (A) strand contains coding domains of significant size (Shapira et al., 1991). The long open reading frame, designated ORF A, is preceded by a 495 nucleotide (nt) noncoding leader sequence and extends 1,869 nt. The junction between ORF A and ORF B, is contiguous with ORF A and extends 9,498 nt. The junction between ORF A and ORF B consists of the pentanucleotide 5'UAAUG-3' in which the UAA portion serves as a termination codon for ORF A (Choi et al., 1991a) and the AUG portion is the 5'-proximal initiation codon of ORF B (Shapira et al., 1991). An 851 nt 3' noncoding domain follows ORF B, terminating with the 3'-poly (A) tail.
ORF A encodes two polypeptides, p29 and p40, that are released from a polyprotein, p69, by an autocatalytic event mediated by p29 (Choi et al., 1991a). Cleavage occurs between Gly -248 and Gly-249 during translation and is dependent upon the essential residue Cys-162 and His-215 (Choi et al., 1991b). Expression of ORF B also involves an autoproteolytic event in which a 48 kDa polypeptide, designated p48, is released from the N-terminal portion of the encoded polyprotein (Shapira et al., 1991). Cleavage of p48 occurs between Gly-418 and Ala0419 and is dependent upon essential residues Cys-341 and His-388 (Shapira and Nuss, 1991). Both p29 and p48 resemble papain-like proteases. Putative RNA-dependent RNA polymerase and RNA helicase motifs have been located in the C-terminal half of ORF B (Shapira et al., 1991; Koonin et al., 1991).
Efforts to determine the precise genetic information responsible for transmissible hypovirulence have been hampered by the inability of HAV RNAs to initiate an infection by an extracellular route. This is a common property of mycoviruses and unencapsidated viral-like RNAs (Buck, 1986; Wickner 1989; El-Sherbeini and Bostian, 1987). We were recently able to partially overcome this limitation by introducing HAV coding domains into virus-free virulent C. parasitica strains by DNA-mediated transformation (Choi and Nuss, EMBO J. 1992). Significantly, transformation with a cDNA copy of the first L-dsRNA open reading frame, ORF A, conferred a number of traits similar to those exhibited by the- L-dsRNA-containing hypovirulent strain (EP713). These traits included reduced pigmentation, reduced laccase accumulation and suppressed conidiation, characteristics that frequently, but not universally, accompany hypovirulence-associated traits- (listed in Hillman et, al. 1990). However, virulence was not reduced in the ORF A transformants. These results established a direct cause and effect relationship between the viral ds RNA present in a hypovirulent c. parasitica strain and specific traits associated with that strain. The fact that these hypovirulence associated traits were conferred in viral-free transformants demonstrated that they are not the result of some general, reaction, of the fungus to the physical presence of replication viral RNA but are caused by a specific coding domain. The observation that reduced virulence was uncoupled from associated traits such as suppressed sporulation in the ORF A transformants suggests that different viral encoded proteins may be responsible for specific traits expressed by individual hypovirulent strains.